RBD-induced neurite sprouting is blocked in primary sensory neurons pre-treated with a Src-family kinase inhibitor, PP2.

<p><b>A</b>, Phase-contrast images of cultured sensory neurons after 2 weeks of treatment. Note the reduction of sprouting in RBD (100 nM) treated cultures, but not NGF (50 ng/ml) treated cultures with treatment of PP2 (1 µM) for 30 min prior to the addition of factors. Scale bar, 50 µm. <b>B</b>, Levels of GAP-43 mRNA in primary cultures treated with RBD (100 nM) or NGF (50 ng/ml) for 2 weeks, with or without PP2 treatment. *<i>p</i><0.05 (n=3-4 independent experiments).</p

Structurally diverse LRP1 ligands such as MMP-9-PEX, but not mutated RBD (muRBD), promoted sensory neurite extensions.

<p><b>A</b>, Phase contrast images of cultured sensory neurons treated with the hemopexin domain of MMP-9 (MMP-9-PEX; 100 nM), mutated RBD (muRBD; lysine residues 1370 and 1374 that are essential for LRP1 binding are replaced by alanine; 100 nM) or GST (100 nM). Scale bar, 50 µm. <b>B</b>, Immunofluorescence microscopy of β-neuronal class III tubulin (Tuj1; green) in primary DRG neurons cultured for 2 weeks in MMP-9-PEX or GST. Scale bar, 10 µm. Cell nuclei are identified by Dapi (blue). Images represent n=2 independent replicates. <b>C</b>, GAP-43 mRNA levels in cultured sensory neurons treated with GST, MMP-9-PEX or muRBD every other day for 2 weeks. Results are compared to vehicle-treated control cultures. Data are expressed as mean±SEM (n=2-4). **<i>p</i> < 0.01 compared with controls.</p

Relationship between serum IL-6 levels and survival.

<p>(<b>A</b>) A conservative threshold linked 20 or 21 pg/ml of IL-6 in serum (y-axis) with 21 to 28 days of survival time (x-axis), as determined by AIC analysis (see Methods), satisfactorily identifying the most appropriate cut-off level (z-axis, AIC = −10.395). Note that patients with serum IL-6 levels ≥21 pg/ml were predicted to have less survival time. (<b>B</b>) The probability of surviving one and three months after evaluation was lower in patients with IL-6 >21 pg/ml than those with <21 pg/ml (one month survival, 20.0% vs. 77.8%, p = 0.007; and three months survival 10.0% vs. 33.3%, p = 0.025). (<b>C</b>) The proportion of patients with IL-6 ≥21 pg/ml was 80.0% for patients surviving less than one month, 20.0% for patients surviving one to three months and 25.0% for patients surviving more than three months.</p

RBD promotes neurite sprouting in primary embryonic sensory neurons that is blocked by the LRP1 antagonist, GST-RAP.

<p><b>A</b>, Phase-contrast images of DRG neurons cultured on collagen using an inverted microscope. DRG neurons were pretreated with or without GST-RAP (200 nM) for 30 min prior to the addition of RBD or NGF (50 ng/ml). Images represent n=6 independent studies. Scale bar, 50 µm. <b>B</b>, Immunolabeling of primary embryonic neurons with β neuronal class II tubulin (Tuj1; green) and Dapi (blue). Scale bar, 15 µm. <b>C</b>, Quantification of neurite sprouting in DRG neurons. Data are expressed as mean ± SEM (n=4 independent studies). In each study, 3 replicate wells were used to measure a minimum of 30 axonal extensions. **<i>p</i><0.01 compared with respective GST or RAP controls. <b>D</b>, Immunoblot analysis of ERK1/2 in GST-RAP-treated DRG neurons subsequently stimulated with RBD or NGF for 30 min. The blot represents 2 independent experiments. <b>E</b>, Quantification of neuronal viability after two weeks in primary culture. Data are expressed as mean ± SEM (n=3) (**<i>p</i><0.01).</p

Patients’ characteristics and comparison between patients with or without cachexia.

<p>Plus-minus data are presented as means ± SD.</p

Axonal Receptivity to myelination by Schwann cells after treatment with LRP1 ligands.

<p><b>A</b>, Phase-contrast images of primary sensory neurons treated with NGF (50 ng/ml) or RBD (100 nM) for 2 weeks and now with Schwann cells for 48 hours. Scale bar, 50 µm. Images represent n=3 independent replicates with internal duplicates; <b>B</b>, Levels of the non-compact myelin marker, MAG and <b>C</b>, compact myelin marker, P0 mRNA in Schwann cells after 48 hours of co-culture with RBD or NGF treated DRG neurons. Data are expressed as mean ± SEM. *<i>p</i> < 0.01 compared with GST (n=3 independent experiments).</p

LRP1 promotes survival in primary embryonic sensory neurons and is abundantly expressed in neonatal dorsal root ganglia (DRGs).

<p><b>A</b>, Immunofluorescence microscopy of cleaved caspase-3 (red) and β-neuronal class III tubulin (Tuj1; green) in primary DRG neurons cultured for 2 weeks. Scale bar, 12 µm. Cell nuclei are identified by Dapi (blue). Images represent n=3 independent replicates. <b>B</b>, Quantification of cleaved caspase-3 positive neurons. Neurons in random fields within each treatment group were counted (n=3 independent experiments). <b>C</b>, Immunoblot analysis of LRP1 (85 kDa) in uninjured adult (8 weeks) or neonatal (1 day) rat DRGs. DRGs were solubilized in RIPA buffer supplemented with sodium orthovanadate and proteinase inhibitors. β tubulin was used as a loading control. Equal amounts of cellular protein (20 µg) were loaded into each lane and subjected to SDS-PAGE and electrotransferred to nitrocellulose for detection with specific antibodies. Each lane represents an individual rat. <b>D</b>, Quantification of LRP1 by densitometry. Data are expressed as mean ± SEM (n=5-7 rats), <i>**p</i> < 0.01 compared with respective GST.</p

Effects of MR16-1 on carcass weight (a), tumor growth (b), food intake (c), and water intake (d).

<p>Transplantation of IL-6-expressing LLC cells caused cachexia in mice (groups 3 and 4), while groups 1 and 2 received only the same amount of saline solution, 0.9% sodium chloride (healthy control groups). We administered MR16-1 (groups 2 and 4) or 0.9% saline (groups 1 and 3), respectively. Body weight, tumor size and food and water intake were measured on days 0, 3, 7, 10, 14, 17 and 21 after transplantation. The measured quantity of food or water was divided by the number of mice and days to determine each intake per animal per day. Statistical significance was evaluated with Tukey’s Honest Significant Differences (HSD) after the one-way ANOVA. Data represent mean and S.D. (S.D. data of weight are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102436#pone-0102436-t004" target="_blank">Table 4</a>). Tumor growth was not significantly different between groups 3 and 4 (B), but body weight (A) and food (C) and water intake (D) were significantly improved in group 4 (†, p<0.01). No significant differences were observed in the carcass weight and food and water intakes between the untreated (group 1) and treated healthy mice (group 2).</p

Safety of MR16-1 in healthy control mice.

<p>To evaluate the safety of MR16-1, we appraised other biochemical parameters in the blood of healthy control mice. Group 2 received MR16-1 treatment, whereas group 1 received the same amount of saline solution.</p><p>Abbreviations used are: AST, aspartic acid aminotransferase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase.</p

Log–log plots of representative cumulative frequency distributions of the size of LAA clusters.

<p>In subjects with COPD (A), the five lung slices analyzed had LAA% values of 2.2 (●), 5.0 (〇), 15.2 (▲), 28.0 (Δ), and 42.1 (◆). In these five slices, <i>r</i>^<i>2</i> has a value of 0.96, 0.98, 0.99, 0.98, and 0.99, respectively. The corresponding D values are 0.97, 1.12, 0.98, 0.96, and 0.83, respectively. In subjects with LAM (B), the five lung slices analyzed had LAA% values of 2.0 (●), 6.7 (〇), 16.7 (▲), 26.9 (Δ), and 40.9 (◆). In these five slices, <i>r</i>^<i>2</i> has a value of 0.96, 0.93, 0.98, 0.95, and 0.89, respectively. The corresponding D values are 1.24, 1.34, 1.27, 1.11, and 1.11, respectively. In subjects with BHDS (C), the five lung slices analyzed had LAA% values of 2.0 (●), 5.7 (〇), 15.9 (▲), 24.6 (Δ), and 40.2 (◆). In these five slices, <i>r</i>^<i>2</i> has a value of 0.72, 0.84, 0.97, 0.99, and 1.0, respectively. The corresponding D values are 0.74, 0.78, 0.76, 1.30, and 0.97, respectively.</p> <p>BHDS: Birt-Hogg-Dubé syndrome; COPD: chronic obstructive pulmonary disease; LAA: low attenuation areas; LAA%: percentage of lung field occupied by low attenuation areas; LAM: lymphangioleiomyomatosis.</p